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ADP-Glo™ Max Assay

The ADP-Glo™ Max Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure ATPase or kinase activity by quantifying the amount of ADP produced in a reaction. The assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) when higher ATP concentration is required (up to 5mM). The ADP-Glo™ Max Assay produces a strong signal that positively correlates with enzyme activity and can be adapted to a multitude of plate formats.

The assay is performed in two steps: first, after the completion of the ADP...

The ADP-Glo™ Max Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure ATPase or kinase activity by quantifying the amount of ADP produced in a reaction. The assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) when higher ATP concentration is required (up to 5mM). The ADP-Glo™ Max Assay produces a strong signal that positively correlates with enzyme activity and can be adapted to a multitude of plate formats.

The assay is performed in two steps: first, after the completion of the ADP-producing reaction, an equal volume of ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP. Second, the ADP-Glo™ Max Detection Reagent is added to simultaneously convert ADP to ATP, and the latter is converted to light in a coupled reaction with luciferase/luciferin.

The ADP-Glo™ Max Assay has a high dynamic range and produces a strong signal at low ATP to ADP conversion, making it well suited for screening low-activity ATPases such as drug membrane transporters and heat shock proteins. The assay produces minimal false hits and Z´ values of greater than 0.7.

Applications

Notes

The ADP-Glo™ Max Assays contain enough reagent to perform the indicated number of reactions in 384-well format using 5μl, 5μl and 10μl of an enzyme reaction, ADP-Glo™ Reagent and ADP-Glo™ Max Detection Reagent, respectively, per sample.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

Storage Conditions

Store the system at –20°C. Before use, thaw all components completely at room temperature. Once thawed, mix all components thoroughly before use. Because ATP is naturally prone to hydrolysis after freeze-thaw cycles dispense into single-use aliquots and store at –20°C. Once prepared, dispense, ADP-Glo™ Max Detection Reagent (ADP-Glo™ Max Detection Buffer + Substrate) into aliquots and store at –20°C. ADP-Glo™ Max Detection Buffer may form a precipitate when thawed. See Section 3.A of the Technical Manual for a protocol to dissolve any precipitate. For convenience, ADP-Glo™ Reagent and ADP-Glo™ Max Detection Reagent may be kept at room temperature (22°C) for 24 hours without loss of signal.

For product intended use please see Patents & Disclaimers tab.

Figure 1. Principle of the ADP-Glo™ Max Assay.

The assay is performed in two steps: 1) after the ATPase or kinase reaction, ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP; and 2) the ADP-Glo™ Max Detection Reagent is added to convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. The light generated correlates to ADP present and ATPase activity.

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